Characterization of dimeric forms of human pituitary growth hormone by bioassay, radioreceptor assay, and radioimmunoassay

Abstract

Seven highly purified dimeric forms of human pituitary growth hormone, composed of the monomeric forms 20 K hGH, 22 K hGH and 24 K hGH linked together by noncovalent or covalent bonds, have been characterized by an in vitro bioassay (the Nb2 assay), a radioreceptor assay and a radioimmunoassay. Considerable differences in the ability to displace labelled recombinant hGH were observed in the radioreceptor assay. The seven dimeric forms varied over a range between 22 K hGH (most effective) and 20 K hGH. The three covalently-linked dimeric forms had nearly identical affinity constants. The mitogenic action of all but one of the hGH dimers in the Nb2 assay was in the same mutual order as the receptor binding activity in the radioreceptor assay. In the RIA, all dose-response curves were parallel except for those obtained with 20 K hGH and with the dimeric form (20 K-20 K)hGH. In this assay, dimeric variants of the constituents 22 K hGH and 24 K hGH were approximately twice as active as 22 K hGH on a molar basis, suggesting about the same affinity between the antibodies and each of the monomeric forms. Determination of the amino acid compositions of the dimeric forms provided support for their content of monomeric constituents as established earlier by electrophoretic analysis.

Growth Hormone Immunoreactivity Does Not Reflect Bioactivity

“…Another possible explanation of our results is that the two assays might recognize different dimers of GH (25). It has been shown, in fact, that an acidic 20-kD/22-kD heterodimer of GH is 2.5 times more potent than the 22-kD molecule alone in stimulating Nb2 cells, although these two variants show a similar immunoreactivity (26).…”

An Investigation into the Lability of the Bioactivity of Human Growth Hormone Using the ESTA Bioassay

“…Although there have been several reports that hGH dimers have a lower affinity for some GH receptors and are less bioac tive than the monomers when tested in a variety of sys tems [10][11][12], Brostedt et al [12] also described the for mation of a noncovalent 20-to 22-kD heterodimer which was 2.5-fold more potent than 22 kD hGH in an Nb2 cell bioassay, but not in an immunoassay run in parallel. Such dimers and isoforms of hGH might be expected to have delayed clearance rates [13][14][15], and may therefore tran siently accumulate in samples taken from secretory pro files in response to provocation [16][17][18][19].…”

High Molecular Weight Growth Hormone ( > 160 kD) in Human Serum Characterized with Monoclonal Antibodies

“…The protein concentrations of the hGH variants have previously been determined spectrophotometrically [26],…”